Cannabinoid receptors (CB/1 and CB/2) and endogenous agonists for these receptors (anandamide and 2-arachi-donoyl glycerol) together form the "endogenous cannabinoid system". As stable analogs of endogenous cannabinoids should have clinical applications and/or serve as powerful experimental tools with which to explore the physiology of this system, part of the project will be directed at developing such agents. This will be achieved by using analogs specially designed and synthesized for this project, to explore the structural features of endogenous cannabinoids that govern their potency in systems that contain the cannabinoid receptor type found on central and peripheral neurons (CB/1). The project will also establish the structural features of endogenous cannabinoid analogs that determine their efficacy at CB/1 receptors and compare these with those determining their affinity for CB/1 receptors. One practical outcome of this exercise will be the development of endogenous cannabinoid analogs whose potencies depend mainly on efficacy. Such compounds are expected to have more consistent pharmacological properties in different biological systems than agonists whose potency depends mainly on affinity. The other main objective of this project is to determine the basis of cannabinoid tolerance by establishing the extent to which tolerance induced by in vivo administration of a CB/1 receptor agonist can be attributed to decreases in CB/1 receptor affinity, CB/1 receptor density/internalization and/or CB/1 coupling efficiency. Guinea-pig or mouse isolated tissue preparations that contain CB/1 receptors on nerve terminals will be used as functional bioassay systems to provide potency values and to investigate tolerance, induced by in vivo cannabinoid treatment. Binding assays will be used to determine CB/1 receptor affinity values and also to detect any changes in cannabinoid receptor affinity or density associated with tolerance. It is intended to monitor CB/1 receptor coupling efficiency in tolerant and non- tolerant tissues by measuring the ability of CB/1 receptor agonist to enhance GTPgammaS binding and to look for receptor internalization by immunofluorescence confocal microscopy.